Radioimmunoassay was developed in 1960 by Yarlow and Berson as a method for detecting or quantitating antigens or antibodies using radiolabeled reactants. Since the initial studies in 1960, radioimmunoassay (RIA) has developed into a versatile analytical technique, particularly useful in clinical laboratories to quantitate a wide variety of compounds. With RIA, the unknown concentration of an unlabeled antigen is determined by comparing its inhibitory effect on the binding of a radioactively-labeled antigen to an antibody. RIAs do have a number of significant limitations, however, including a limited shelf-life, high cost, and potential environmental harm.
The disadvantages associated with RIAs led to the development of the enzyme immunoassay (EIA), in which the activity of an enzyme is measured to quantitate an analyte. EIAs are subdivided into heterogeneous assays and homogeneous assays. Heterogeneous EIAs require a physical separation of the antibody-bound, labeled analyte from the unbound labeled analyte. With homogeneous ElAs, a separation step is not required. Homogeneous EIAs have been successful commercially because of their speed, simplicity, and automation. The enzymatic activity associated with EIAs is often monitored spectrophotometrically, using a substrate which produces a unique chromophore as a result of an enzymatic reaction.
In addition to using spectrophotometric detection techniques, EIAs have been developed which use electrochemistry to monitor activity of the enzyme label. With electrochemical detection, the active enzyme causes the formation of an active electron mediator or a redox couple from an inactive substrate. The activated mediator or redox couple then shuttles electrons from the enzyme to the electrode or from the electrode to the enzyme. The resulting current can be measured and correlated to analyte level.
Direct electrochemical enzymatic assays (non-immunological) are also known in which the presence or absence of the analyte to be measured causes an electroactive compound to be cleaved from a non-electroactive substrate. The electroactive compound may then be oxidized or reduced and the resulting current measured.
Enzyme complementation immunoassays have also been developed, such as CEDIA.RTM. (Cloned Enzyme Donor ImmunoAssay--a registered trademark of the Microgenics Corporation) technology, an example of which is described in U.S. Pat. No. 4,708,929 (issued Nov. 24, 1987), which is hereby incorporated by reference. CEDIA.RTM. technology involves the use of enzyme acceptor and enzyme donor polypeptides prepared by recombinant DNA techniques or synthetic peptide synthesis techniques which are capable of spontaneously associating in solution to form an active enzyme complex. This association can be modulated, for example, by conjugating the enzyme donor polypeptide to a member of a specific binding pair, and providing the complimentary member of the specific binding pair elsewhere in the assay. The enzyme donor polypeptide may also be chemically modified to include a specific recognition site that is not a member of a specific binding pair (e.g., a protease site or an esterase site). Accordingly, in its broadest sense, CEDIA.RTM. technology allows the formation of an active enzyme complex by the spontaneous association of enzyme acceptor and enzyme donor polypeptides to be dependent on the presence or concentration of an analyte of interest. The amount of enzymatic activity is then monitored spectrophotometrically.
One embodiment of CEDIA.RTM. technology is shown in FIG. 1. Analyte analog 1 is covalently attached to enzyme donor polypeptide 2 to form enzyme donor polypeptide conjugate 3. Analyte-specific antibody 4 can be used to inhibit reassembly of enzyme donor polypeptide conjugate 3 with enzyme acceptor polypeptide 6. When a sample containing analyte 8 is introduced, analyte 8 and enzyme donor polypeptide conjugate 3 compete for binding to antibody 4. As the amount of analyte 8 increases, less enzyme donor polypeptide conjugate 3 binds to antibody 4 and more active enzyme 10 is formed. Active enzyme 10 hydrolyzes enzyme substrate 11 (e.g., chlorophenol-red-.beta.-D-galactopyranose (CPRG)), which then undergoes a color change and is monitored spectrophotometrically.